It is well known (e.g. in "Papilloma Viruses and Human Cancer" edited by H. Pfister and published by CRC Press Inc. in 1990) that papilloma viruses can be classified into several distinct groups based on the host in which they infect. Human papilloma viruses (HPV) can be further differentiated into types 1-56 depending on DNA sequence homology. Types 16, 18 and 42 are associated with the majority of in situ and invasive carcinomas which may occur in the anogenital tract and in particular the cervix. In this regard, a number of cervical intra epithelial neoplasias and carcinomas of the cervix have been associated with HPV16 and HPV18. (Lancaster et al 1987: Cancer Metast. Rev. 6653 and Pfister 1987. Adv. Cancer Res 48 113). These same two references also point out that papilloma viruses are small DNA viruses encoding up to 8 early and 2 late genes.
The protein from the expression of the early gene E7 varies between 93 to 127 amino acid residues. The E7 protein is the most abundant viral protein in HPV16 containing CaSki and SiHa squamous carcinoma cell lines and in HPV18 containing HeLa and C4-1 lines. (Seedorf et al. 1987. EMBO J. 6, 139). DNA transfection experiments implicate the E6 and E7 ORF proteins in in vitro transformation of mouse fibroblasts (Yasumoto et al. 1986. J. Virol., 57, 572), rat epithelial cells (Matlashewski et al. 1987. EMBO J. 6, 1741) and primary human keratinocytes (Schlegel et al. 1988, EMBO J. 7, 3181; Pirisi et al. 1987. J. Virol. 61, 1061). Cooperation with an active ras oncogene leads to full transformation (Matlashewski et al. 1987. EMBO J. 6, 1741) and there is a requirement for continued expression of the E7 gene to maintain the transformed phenotype (Crook et al. 1989. EMBO J. 8, 513). The E7 protein may be recognised by the immune system, since anti E7 antibodies can be detected in the serum of approximately 20% of patients with HPV16 associated cervical lesions (Jenison et al. 1988. J. Virol. 62, 2115; Jochmus--Kudielka et al. 1989. J. Natl. Cancer Institute 81, 1698; Smillie et al. 1990. Immunol. Infect. Dis. 1, 13).
In addition to types 16, 18 and 42, further genotypes 6, 11, 31, 33, 35 and 39 which fall within the same sub group as types 16, 18 and 42 are infective for anogenital epithelium (Gissman Cancer Surveys 3. 162-181 (1984) Zur Hausen & Schneider The Papillomaviruses p245-263 Edited by Howley and Salzman New York Plenum Press (1987)). The DNAs of HPV types 16 and 18 are frequently found in genital tumours (Durst et al.) J. Gen Virol. 66 1515-1522 (1985), Gissman et al PNAS 80, 560-563 (1983)) supporting the concept that members of this sub group have an essential role in the etiology of genital cancer (Syrjanen et al British Journal of Obstetrics and Gynaecology 92 1086-1092 (1985)). Integration of HPV16 DNA into host genomes is frequently observed in cervical cancers with interruption of the E2 viral ORF, the protein product of which region transregulates early ORF transcription from the p97 promoter and retention of intact E6 and E7 ORFs.
Abundant circumstantial evidence implicates host immune mechanisms in the control of HPV associated tumours of the anogenital epithelium (Singer et al British Medical Journal 288, 735-736 1984). There is an increased incidence of pre-neoplastic (Frazer et al Lancet ii 657-660 1986) and neoplastic associated lesions in homosexual men immunosuppressed by human immunodeficiency virus infection and a markedly increased risk of squamous cell carcinoma (SCC) of the cervix and vulva but not of control organs such as breast and rectum in immunosuppressed allograft recipients (Sheil and Flavel Ninth Report of Australian and New Zealand Combined Dialysis and Transplant Registry pp 104-112 Edited by APS Disney 1986).
Taken with the above, the normal natural history of HPV infection in most patients with alpha-gamma globulinemia suggests that cellular rather than humoral responses are important for the control of the phenotypic expression of HPV infection (Kirschner Progress in Medical Virology 1986).
Standard immunological approaches to the study of anogenital HPV infection have been hampered by the lack of a suitable animal model and of an in vitro epithelial cell culture permissive for HPV.
Vaccines have also been proposed in regard to HPV with however only indifferent success.
It has been proposed to use vaccines containing autogenous tumor homogenates (Abcarian et al J. Surg Res 22: 231-236 (1977) Dis Colon Rectum 25:64851 1982 Dis Colon Rectum 19: 237-244 (1976)). However it has recently been advocated that patients should no longer be treated with autogenous vaccines because of the potential oncogenic effect of the viral DNA (Bunney 1986 Br Med J 293 1045-1047).
In relation to production of genetically engineered vaccines this matter has been discussed in Pfister (1990) above and it seems that difficulty has been experienced in obtaining an effective vaccine because of the plethora of different papilloma virus types. Pfister however points out that attention should be directed to the so called early proteins (ie. E1, E2, E3, E4, E5, E6, E7 or E8) because these proteins are most likely synthesised in the proliferating basal cells of a wart infection in contrast to the structural proteins which are expressed in the upper epidermal layers. Therefore according to Pfister (1990) virus capsid protein appears to be limited in relation to use in a vaccine. The use of recombinant vaccinia viruses in in vitro test systems for papilloma virus early proteins in eukaryotic cells has been discussed also in Pfister (1990). This may take the form of a live vaccine consisting of genetically modified vaccinia virus expressing papilloma virus proteins or on the surface of paraformaldehyde fixed autologous cells infected in vitro with vaccinia recombinants or transfected with other expression vectors. Another strategy for vaccine development as discussed in Pfister (1990) is to use an immune stimulating complex of the glycoside
Data on successful prophylactic vaccination exist only for bovine fibropapillomas homogenised homogenate of bovine fibropapillomas and has been shown to provide limited immunity (Olson et al. J am Vet Med Assoc 135, 499 (1959) Cancer Res 22 463 (1962)). A vaccine including an engineered L1 fusion protein (Pilacinski et al. UCLA Symp. Molecular and Cellular Biology New Series Vol 32 Papilloma Viruses Molecular and Clinical Aspects Alan R Liss New York 1985 257) has also been used in calves but proved unsuccessful in humans. In Pfister (1990) it is stated that there is presently no evidence for a possible prevention of HPV infection by the use of a capsid protein vaccine, but induction of an antitumor cell immunity appears to be feasible.
The L1 and L2 genes have been the basis of vaccines for the prevention and treatment of papilloma virus infections and immunogens used in the diagnosis and detection of papilloma viruses (International Patent Specifications W08605816 and E08303623). However, it appears that no commercial usage of these vaccines have taken place.
Reference may also be made to Patent Specification EP386734 which describes new immunogenic regions of HPV16 E7 protein which may be useful in vaccines, EP 375555 which describes HPV16 peptides useful as immunoassay reagents for the detection of HPV16 proteins and which contain an antigenic determinant for HPV16, a reference in VACCINE (1990) 8 3, 199-204 which describes vaccines including recombinants expressing HPV E5, E6 or E7 ORF intended for use in providing antitumor activity, Australian Specification 52860/90 which describes screening antibodies for specificity to an antigen which is an epitope of HPV16 L1 or E7 proteins, Australian Specification 75535/87 which describes synthetic peptides of HPV corresponding to an amino acid sequence region having at least one reverse turn and predicted hydrophilicity, Patent Specification EP217919 which describes type specific papillomavirus DNA sequences and peptides useful in vaccines containing 15-75 nucleotides, U.S. Pat. No. 4,551,270 which describes at least one antigenic determinant of papillomavirus and immunogens and vaccines containing the antigenic determinant, Patent Specification EP412762 which describes a polypeptide having the sequence Leu-Tyr-Cys-Tyr-Glu-Gln-Leu-Asn-Asp-Ser-Ser (SEQ ID NO:51) which inhibits binding of the HPV E7 protein to retinoblastoma gene which may be used in vaccines for treatment of cervical cancer and genital warts, French Specification 2643817 which describes a vaccine for treatment of tumours induced by papillomavirus containing recombinant poxvirus with heterologous DNA encoding region of non structural papillomavirus, Japanese Specification J01061665 which describes an antibody formed to an antigen polypeptide of HPV16 E6 or E7 protein which antigen polypeptide is Tyr-Gln-Asp-Pro-Gln-Glu-Arg-Pro-Arg-Lys-Leu-Pro-Gln-Leu-Cys (SEQ ID NO:52) which is part of E6 protein or Cys-Tyr-Gln-Leu-Asn-Asp-Ser-Ser-Glu-Glu-Asp-Glu-Ile-Asp (SEQ ID NO:53) which is part of E7 protein, Australian Specification 76018/87 which describes expression products of HPV16 or HPV18 which may be used for the production of antibodies, EP235187 which describes kits containing polypeptide(s) expressed by several groups of papilloma virus including HPV16 and HPV18 which are expression products of E6, E7 or L2 genes and U.S. Pat. No. 4,777,239 which includes, diagnostic synthetic peptides for HPV one of which includes residues 45-58 of protein E6 and 40-50 of protein E7 which may be used as a therapeutic agent.
Of particular interest in the prior art discussed above is specification EP375555 which describes a peptide AEPDRAHYNIVTFC (SEQ ID NO:56) which may be used as an immunoassay reagent for diagnosis of HPV16 antibodies. This peptide includes the DRAHYNI (SEQ ID NO:11) sequence. However it is clear from a review of this document that there was no realisation that the DRAHYNI (SEQ ID NO:11) sequence corresponded to a T helper cell epitope of the ORF of E7 protein of HPV16 and the consequences in regard to HPV therapy as discussed in this patent specification.
Of particular relevance also is specification EP386734 which discloses a number of peptides, one of which (i.e., No. (V)) comprises the sequence Asp-Glu-Ile-Asp-Gly-Pro-Ala-Gly-Gln-Ala-Glu-Pro-Asp-Arg-Ala-His-Tyr (SEQ ID NO:54). It will be noted that this sequence includes the sequence DRAHY (SEQ ID NO:55). While this particular peptide is described as corresponding to a useful immunogenic region of HPV16 E7 protein, and thus useful in vaccines, it will be appreciated from the discussions hereinafter that the sequence DRAHYNI (SEQ ID NO:11) has a more useful antigenic property and thus will stimulate a far greater immune response.